We are studying the molecular basis for corneal-specific gene expression. Mouse aldehyde dehydrogenase 3a1 (ALDH3A1) comprises half of the water-soluble protein in the corneal epithelial cells of the mouse. Cytoplasmic corneal proteins at such high levels have been called corneal crystallins. We have described the developmental expression of the mouse Aldh3a1 gene. Corneal expression was first detected after birth and high levels were found at postnatal day 14, the time of eye opening and stratification of the epithelial cells. Pax6, Oct1 and p300 were found to synergistically activate the ALDH3A1 promoter. In addition, Sox1, c-maf and KLF5 activate the Aldh3a1 promoter.[unreadable] [unreadable] We previously reported that a gelsolin-like protein comprises half of the water-soluble protein of the zebrafish cornea. We have now shown that this corneal crystallin is scinderin-like a (scinla), and that another family member (scinlb) is also preferentially expressed n the cornea at lower levels. The two genes have almost identical exon-intron structures and encode proteins with 72% identity, but have different developmental expression patterns. scinla is expressed highly in the cornea already by 1 day after fertilization, but scinlb is not expressed in the cornea even at 6 days of development. Promoter activities of the two genes mimicked their gene expression patterns in transgenic zebrafish. Specific reduction of the expression of each gene with morpholino oligonucleotides gave different phenotypes. It thus appears that scinla and scinlb have evolved different biological functions due to different expression patterns during zebrafish development.[unreadable] We are examining the ocular phenotypes of transgenic mice overexpressing Pax6 and Pax6(5a) transgenes in the cornea. The overexpressing corneas are opaque and enriched with blood vessels. Further studies involving microarray analysis are in progress to identify downstream gene targets of the Pax6 transcription factors. [unreadable] [unreadable] In the previous fiscal year, KLF4, the most abundant corneal transcription factor identified by SAGE analysis, was deleted specifically in the mouse cornea (Klf4 conditional knockout, or Klf4CN). The Klf4CN mice were microphthalmic with defects in lens, cornea, iris and conjunctiva. Expression of the keratin-12 and aquaporin-5 genes was down regulated, consistent with Klf4CN corneal epithelial fragility and stromal edema, respectively. We showed this year that the promoter of the Slurp-1 gene binds and is activated by KLF4 in corneal epithelial and, especially, in skin keratinocyte cells. We also showed increased collagen interfibrillar spacing and possibly changes in fibril diameter and collagen-proteoglycan associations in the cornea of Klf4CN mice. We have identified diverse potential KLF4 target genes by microarray analysis, revealing the molecular basis of the wide range of Klf4CN corneal phenotypes. Overall, these results establish KLF4 as an important node in the genetic network of transcription factors regulating corneal homeostasis. These observations provide new insights into the role of KLF4 in post-natal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann dystrophy and Stevens-Johnson syndrome.[unreadable] [unreadable] Finally, we continued our studies on microRNAs in the cornea. miRNAs are a class of small endogenous noncoding RNAs that posttranscriptionally down regulate gene expression and play important roles in embryonic development, differentiation, and cancer. Microarray analysis in post-natal day 9 (PN9) and 6-week-old (adult) mice showed that miRNAs display dynamic changes in miRNAs after eye opening and epithelial stratification in the cornea. These changes were consistent with the possibility that they play important roles in the regulating cell proliferation and differentiation of the cornea. miRNA184 was found especially enriched in the cornea. 78 of the 568 miRNAs showed differential expression between the PN9 and 6-week-old mouse cornea. Conditional deletion of Dicer, which is the enzyme responsible for miRNA maturation, in the lens and cornea resulted in microphthalmia, loss of lens structure, convolution of the retina and absence of anterior/posterior and vitreal chambers. With respect to the cornea, the conditional Dicer knockout mice showed a loss of the corneal endothelium and an absence of corneal epithelial stratification. We conclude that miRNAs play a critical role in eye and corneal development.